It has been proposed that the mechanism of the vasodilator action of glyceryl trinitrate (GTN) involves biotransformation to nitric oxide. A sensitive chemiluminescence method for nitric oxide determination was used to test this hypothesis. In four experiments, bovine pulmonary artery (BPA) was incubated with GTN (0.1 mM) in Krebs' solution (2 mL) containing 30 mM KCl, and in anaerobic conditions using 95% Ar – 5% CO2, in a sealed micro-Fernbach flask (6.2-mL volume). After incubation for 2, 5, 10, or 20 min at 37 °C, 400-μL aliquots of headspace gas were removed and injected into a redox chemiluminescence detector. Nitric oxide formation was first measurable at 5 min (76 ± 53 pmol/g wet wt. BPA), and increased with incubation time (174 ± 46 pmol/g wet wt. BPA after 10 min and 310 ± 67 pmol/g wet wt. BPA after 20 min). This is the first direct chemical measurement of nitric oxide formation during interaction of GTN with vascular smooth muscle. These data support the concept that GTN is a nitrovasodilator prodrug acting via the formation of nitric oxide.Key words: nitric oxide formation, glyceryl trinitrate, chemiluminescence, bovine pulmonary artery, vasodilation.